1. Field of the Invention
The present invention is directed to novel mutant cellulase compositions which have improved performance, such as, for example in surfactants known to be problematic when used in conjunction with such a cellulase or under conditions of thermal stress. More specifically, the present invention relates to mutations in EGIII produced by Trichoderma reesei, which mutations provide improved performance under conditions of thermal or surfactant mediated stress.
2. State of the Art
Cellulases are enzymes which are capable of hydrolysis of the .beta.-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and .beta.-glucosidases (Knowles, J. et al., (1987), TIBTECH 5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Primary among the applications that have been developed for the use of cellulolytic enzymes are those involving degrading (wood)cellulose pulp into sugars for (bio)ethanol production, textile treatments like `stone washing` and `biopolishing`, and in detergent compositions. Thus, cellulases are known to be useful in the treatment of mechanical pulp (see e.g., PCT Publication No. WO 92/16687). Additionally, cellulases are known to be useful as a feed additive (see e.g., PCT Publication No. WO 91/04673) and in grain wet milling.
Of primary importance, however, cellulases are used in the treatment of textiles, i.e., in detergent compositions for assisting in the removal of dirt or grayish cast (see e.g., Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826 which illustrate improved cleaning performance when detergents incorporate cellulase) or in the treatment of textiles prior to sale to improve the feel and appearance of the textile. Thus, Great Britain Application No. 1,358,599 illustrates the use of cellulase in detergents to reduce the harshness of cotton containing fabrics and cellulases are used in the treatment of textiles to recondition used fabrics by making their colors more vibrant (see e.g., The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report, Vol. 24, pp. 54-61 (1986)). For example, repeated washing of cotton containing fabrics results in a grayish cast to the fabric which is believed to be due to disrupted and disordered fibrils, sometimes called "pills", caused by mechanical action. This greyish cast is particularly noticeable on colored fabrics. As a consequence, the ability of cellulase to remove the disordered top layer of the fiber and thus improve the overall appearance of the fabric has been of value.
Thus, cellulases have been shown to be effective in many industrial processes. Accordingly, there has been a trend in the field to search for specific cellulase compositions or components which have particularly effective performance profiles with respect to one or more specific applications. In this light, cellulases produced (expressed) in fungi and bacteria have been subject of attention. For example, cellulase produced by certain fungi such as Trichoderma spp. (especially Trichoderma longibrachiatum) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes. For example, Wood et al., "Methods in Enzymology", 160, 25, pages 234 et seq. (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG "), and .beta.-glucosidases (EC 3.2.1.21) ("BG"). The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. U.S. Pat. No. 5,475,101 (Ward et al.) discloses the purification and molecular cloning of one particularly useful enzyme called EGIII which is derived from Trichoderma longibrachiatum.
PCT Publication No. WO 94/14953 discloses endoglucanases which are encoded by a nucleic acid which comprises any one of a series of DNA sequences, each having 20 nucleotides.
Ooi et al., Curr. Genet., Vol. 18, pp. 217-222 (1990) disclose the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus which contains the amino acid strings NNLWG, ELMIW and GTEPFT. Sakamoto et al., Curr. Genet., Vol. 27, pp. 435-439 (1995) discloses the cCNA sequence encoding the endoglucanase CMCase-1 From Aspergillus kawachii IFO 4308 which contains the amino acid strings ELMIW and GTEPFT. Ward et al., discloses the sequence of EGIII having the amino acid strings NNLWG, ELMIW and GTEPFT. Additionally, two cellulase sequences, one from Erwinia carotovara and Rhodothermus marinus are disclosed in Saarilahti et al., Gene, Vol. 90, pp. 9-14 (1990) and Hreggvidsson et al., Appl. Environ. Microb., Vol. 62, No. 8, pp. 3047-3049 (1996) which contain the amino acid string ELMIW. However, none of these references discloses or suggests that these amino acid strings have any particular relevance in identifying or isolating other cellulases, and particularly fail to suggest that such cellulases are obtainable from such diverse organisms as bacteria, Actinomycetes and other filamentous fungi.
Despite knowledge in the art related to many cellulase compositions having applications in some or all of the above areas, there is a continued need for cellulase compositions which have resistance to certain surfactant compositions generally present in compositions with which cellulases are generally used, i.e., household detergents, stonewashing compositions or laundry detergents. One problem with the prior art cellulases has been the sensitivity of such surfactant compositions, for example to linear alkyl sulfonates (LAS). Because surfactants are ubiquitous in detergents, the susceptibility of cellulases to inactivation from such compounds can be highly disadvantageous to their value in these detergents. Nonetheless, EGIII from Trichoderma reesei, while having excellent resistance to LAS type compounds, may be improved by modifying certain residues identified by the Applicants herein as critical to surfactant resistance.